1. Field of the Invention
This invention relates to a method for hematology analysis, wherein fluorescent dyes are used to distinguish various components of a sample of blood.
2. Discussion of the Art
The CELL-DYN® Sapphire™ automated hematology analyzer, as well as the CELL-DYN® 4000 automated hematology analyzer, both of which are commercially available from Abbott Laboratories, Santa Clara, Calif., are equipped with an optical bench that can measure multi-angle light scatter and fluorescence, as described in U.S. Pat. Nos. 5,631,165 and 5,939,326, both of which are incorporated herein by reference. Furthermore, U.S. Pat. Nos. 5,516,695 and 5,648,225, both of which are incorporated herein by reference, describe a reagent suitable for lysing red blood cells and staining nuclear DNA of membrane lysed erythroblasts to discriminate white blood cells from erythroblasts. Membrane lysed erythroblasts are erythroblasts wherein the membrane thereof has undergone lysis. U.S. Pat. No. 5,559,037, incorporated herein by reference, describes the simultaneous detection of erythroblasts and white blood cell differential by means of a triple triggering circuitry (AND/OR), which is used to eliminate noise signals from cell debris, such as, for example, membranes of lysed red blood cells, which are located below the lymphocyte cluster along the Axial Light Loss (ALL) axis of a cytogram. However, the use of lysing agents to lyse red blood cells brings about certain difficulties and complications in the detection of red blood cells and white blood cells. The lysing agent may be insufficiently strong, thereby resulting in red blood cells being counted as white blood cells. Alternatively, the lysing agent may be excessively strong, thereby resulting in artificially low counts of white blood cells. Different samples require lysing agents of different strengths in order to obtain accurate counts of white blood cells; accordingly, all hematology analyzers currently in use sometimes yield incorrect counts of white blood cells.
In hematological assays aimed at determining parameters from human whole blood, there are two physiological factors that present obstacles to simple, rapid, and accurate determination of cell counts. One factor is that, in typical fresh peripheral human whole blood, there are about 1,000 red blood cells and about 50 platelets for each white blood cell. The other factor is that, while platelets are typically sufficiently smaller than any other cell type to allow discrimination based on size, and most white blood cells are sufficiently larger than either red blood cells or platelets to again allow discrimination based on size, two cell species in particular—red blood cells and lymphocytes, a subtype of white blood cells—typically overlap in size distribution (as well as in their scattering signatures) to a sufficient degree to make discrimination based on size prone to gross error. Therefore, when determining red blood cells mainly by size discrimination, the asymmetry in concentration works in one's favor, since the occasional white blood cell misclassified as a red blood cell will not, generally, affect the overall accuracy of the measured concentration of red blood cells to any appreciable degree; however, the converse is not true, and any unaccounted for interference from red blood cells in determining the concentration of lymphocytes (and, by extension, the overall concentration of white blood cells) would yield very inaccurate results.
Consequently, methods have been developed in the prior art to handle this large asymmetry and size overlap and still provide useful results in an acceptable time frame. One standard method employed in the prior art has been to separate the blood sample to be analyzed into at least two aliquots, one destined for red blood cell and platelet analysis, and one for white blood cell analysis. The aliquot destined for white blood cell analysis is mixed with a reagent solution containing a lysing reagent that preferentially attacks the membranes of the red blood cells. Partially on account of their loss of hemoglobin through the compromised membrane, and partially on account of their attendant reduction in size, the resulting lysed red blood cells become distinguishable from lymphocytes based on their respective scattering signatures. Another method employed in the prior art involves using nucleic acid dyes to provide a fluorescent distinction between the red blood cells and the white blood cells. White blood cells contain a nucleus containing DNA. When these white blood cells are labeled via a fluorescent label, they can be distinguished from mature red blood cells, whose nuclei have been expelled in the maturation process.
Both of these methods have drawbacks. First of all, the lysing reagent used to dissolve the red blood cells can attack the white blood cells as well, reducing their integrity and eventually dissolving them. This drawback is magnified with fragile white blood cells, which are abnormal on account of some type of pathological condition (such, as, for example, chronic lymphocytic leukemia). Another drawback is attributable to certain types of red blood cells, such as, for example, those found in neonates, and in patients with thalassemia, sickle-cell anemia, and liver disease, which red blood cells are naturally resistant to lysis, and which red blood cells therefore tend to persist as interferents in white blood cell assays involving lysis. In order to reduce the likelihood of either degradation of white blood cells or interference from unlysed red blood cells (either of which would jeopardize the accuracy of the overall white blood cell concentration measurement), a carefully selected combination of a lysing agent, concentration of the lysing agent, control of temperature, and time of incubation must be used. In some cases, the user is offered several test options with different lysing conditions, thereby allowing the user to tailor the assay to the subject patient sample. This tailoring, however, is a complex solution, which additionally either requires prior knowledge of the state of the patient, or must be used as a reflex test following a standard complete blood count (CBC).
Turning to the previously mentioned fluorescence-based approach for discriminating red blood cells from lymphocytes, a major obstacle is the measurement rate. When white blood cells are measured at the same time as red blood cells and platelets, the presence of red blood cells sets an upper limit to the concentration that can be sent through the analyzer without incurring in coincidences at an unacceptably high rate; the dilution ratio used to achieve such concentration, in turn, limits the rate at which white blood cell events are being counted; and in order to obtain the counting precision expected of the analyzer, this relatively low rate of white blood cell event acquisition, in turn, results in long acquisition times. For example, the concept of measuring all of the components of blood from a single sample in one pass was disclosed in U.S. Pat. No. 6,524,858. As noted in that disclosure, the method would be capable of a cycle time of 88 seconds, or about 41 CBC/hr. This throughput is far lower than that achievable by most automated hematology analyzers commercially available today, severely limiting the commercial usefulness of the one pass method.
The CELL-DYN® Sapphire™ hematology analyzer, as another example, presently offers a test selection (requiring yet another aliquot of sample in addition to those used in the red blood cell/platelet assay and in the white blood cell assay) employing a nucleic-acid dye capable of differentiating between red blood cells and lymphocytes. This test selection uses the dye primarily to differentiate between mature red blood cells and reticulocytes, a subset of immature red blood cells that retain dye-absorbing RNA in the cytoplasm. While it would technically be possible to count the white blood cells using this same assay, because they are sufficiently differentiated by fluorescence from either red blood cells or reticulocytes to obtain the desired accuracy, the relatively low concentration of white blood cells in the dilution used makes it an impractical option to achieve the required statistical precision. Such a scheme would require an acquisition time of approximately 75 seconds, limiting throughput to only 48 CBC/hr. Accordingly, although this approach is theoretically feasible, a much higher throughput would be required in order for this approach to become practical commercially.
Although modern five-part differential hematology analyzers are capable of reporting more hematology parameters, and consequently, providing more useful diagnostics information, almost all of them contain sophisticated fluidic systems, reagent systems, and hardware systems in order to facilitate a number of different assays on blood samples of patients. The complex design often results in higher overall costs of hematology analyzers, as well as greater possibility of poor reliability of the hematology analyzers.
Red blood cells and platelets, as well as their associated parameters, are measured by means of impedance or optical methods, following a dilution of the blood sample with diluent. Quantification of hemoglobin requires lysis of red blood cells by means of a mixture of hemoglobin lysing reagent and, in most cases, a diluent. White blood cell count and white blood cell differential analysis rely on a separate reagent or reagents for lysing red blood cells, minimizing red blood cell fragments, and stabilizing white blood cells for differential measurement. Additional reagents, which may contain one or more fluorescent dyes, are needed to allow hematology analyzers to conduct more complete analysis of blood cells, including reticulocytes and nucleated red blood cells.
Therefore, it would be desirable to develop a method for identifying, analyzing, and quantifying the cellular components of a sample of whole blood by means of multi-angle light scatter without the need for lysing red blood cells, complex fluidic systems, complex reagent systems, or complex hardware systems.